Hyaluronic acid derivatives

ABSTRACT

The present invention relates to an injection solution for intra-articular administration for treating arthritic disorders comprising a cross-linked hyaluronic acid derivative wherein part of functional groups of a hyaluronic acid are cross-linked with a cross-linking group to the extent of 0.6 to 15% of cross-linking extent as an active ingredient in an amount having a long-lasting analgesic effect and a pharmaceutically acceptable carrier, and an analgesic composition for suppressing a cartilage degeneration caused by arthritic disorders or a composition for suppressing a cartilage degeneration or an inflammation of synovium caused by arthritic disorders each comprising the cross-linked hyaluronic acid derivative and a pharmaceutically acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.12/448,064, which is a National Stage of PCT/JP2007/073987, filed Dec.6, 2007, which claims the benefit of provisional application 60/868,798,filed Dec. 6, 2006. The disclosures of patent application Ser. No.12/448,064, PCT/JP2007/073987 and 60/868,798 are incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

This invention relates to pharmaceutical products having a long-lastinganalgesic effect, a cartilage protective effect, and ananti-inflammatory effect for a long term by a single injection.

BACKGROUND ART

Osteoarthritis (OA), the clinical syndrome of joint pain and dysfunctioncaused by joint degeneration, affects more people than any other jointdisease. Osteoarthritis is by far the most common joint disorder in theUnited States and throughout the world, and is one of the leading causesof disability in the elderly. Knee OA is a common but often difficultproblem to manage in primary care. Traditional nonsurgical management,consisting of lifestyle modification, physical therapy and pharmacologictherapy, is often ineffective or leaves residual symptoms. An option forsubjects with symptomatic knee OA is a treatment that involves a seriesof intra-articular injections of hyaluronic acid (hereinafter referredto IA-HA). Sodium hyaluronate is a naturally-occurring constituent ofextra cellular matrix, and following intra-articular injection works asa lubricant and shock absorber to relieve pain and improve knee functionin OA. Currently, there are 5 IA-HA commercially available products inthe United States, SUPARTZ®, SYNVISC®, HYALGAN®, ORTHOVISC® andEUFLEXXA. SYNVISC® was the first IA-HA product launched in the US, andthe only aqueous solution of cross-linked derivatives of hyaluronate,others are aqueous solutions of sodium hyaluronate (hereinafter referredto HA-Na). These currently marketed IA-HA products are not apparentdifferenced in effectiveness. Moreover these products utilize a seriesof 3 to 5 injections and in other words do not result in longer termresponses by a single injection. Therefore new IA-HA product requiringfewer injections would be expected to reduce invasiveness and potentialrisk of joint infection and result in longer term responses by a singleinjection.

So far there are many reports about cross-linked hyaluronate with thedifferent cross-linking methods from SYNVISC®, for example cross-linkedhyaluronate produced by using multifunctional epoxy compound as across-linking group (see Patent Literature 1), photo cross-linkedhyaluronate (see Patent Literatures 2 to 4) and intramolecular bridgedhyaluronate without a cross linker (see Patent Literature 5).Cross-linked hyaluronate is being investigated as a minimally invasivetreatment for pain associated with osteoarthritis of the knee insubjects who have failed to respond adequately to conservativenon-pharmacologic therapy and/or simple analgesics, e.g., acetaminophen.For example, it is reported an injected agent for treating jointdisorder which was improved to produce a merit of decreasing number ofdoses by using an agent comprising hyaluronate gel with or withoutcross-linked structure and phospholipids (see Patent Literature 6).

However, many reports about photo cross-linked hyaluronate product aremainly noted synthesis and general properties of the product and doesnot carefully examined available and restrictive conditions andproperties for an unique application and a specific effect on saidcross-linked hyaluronate product. The comparison of antigenicity andirritant property between SYNVISC® and photo cross-linked hyaluronatewas reported on Reference (see Non-Patent Literature 1).

-   Patent Literature 1: JP-B2-5-74571-   Patent Literature 2: U.S. Pat. No. 5,462,976-   Patent Literature 3: U.S. Pat. No. 5,763,504-   Patent Literature 4: U.S. Pat. No. 6,031,017-   Patent Literature 5: JP-A-2003-252905-   Patent Literature 6: JP-A-2002-348243-   Non-Patent Literature 1: “Evaluation of in vivo biocompatibility and    biodegradation of photocrosslinked hyaluronate hydrogels” J. Biomed    Mater Res. A 70:550-559(2004)

SUMMARY OF THE INVENTION Disclosure of the Invention

This invention was developed to solve the problem of the previouslyavailable IA-HA product that the effect of the products does not last orcontinue over several weeks.

That is, the present invention is as follows:

[1] An injection solution for intra-articular administration fortreating arthritic disorders comprising a cross-linked hyaluronic acidderivative wherein part of functional groups of a hyaluronic acid arecross-linked with a cross-linking group to an extent of 0.6% to 15% ofcross-linking extent based on a total number of constituent disaccharideunit of hyaluronic acid as an active ingredient in such an amount thatshows a long-lasting analgesic effect or a long-continued analgesiceffect, and a pharmaceutically acceptable carrier.[2] The injection solution as described in [1], wherein a degree ofsubstitution of the cross-linking group of the cross-linked hyaluronicacid derivative is 3% to 50% based on a total number of constituentdisaccharide unit of the cross-linked hyaluronic acid derivative.[3] The injection solution as described in [1], wherein a degree ofcross-linking of the cross-linked hyaluronic acid derivative is 5% to40% based on a total number of constituent cross-linking group.[4] An injection solution for intra-articular administration fortreating arthritic disorders comprising a cross-linked hyaluronic acidderivative wherein a degree of substitution of a cross-linking group ofthe cross-linked hyaluronic acid derivative is 3% to 50% based on atotal number of constituent disaccharide unit of hyaluronic acid and adegree of cross-linking is 5% to 40% based on a total number ofconstituent cross-linking group, and a pharmaceutically acceptablecarrier.[5] An injection solution for intra-articular administration fortreating arthritic disorders comprising a cross-linked hyaluronic acidderivative wherein a degree of substitution of a cross-linking group is3% to 50% based on total number of constituent disaccharide unit ofhyaluronic acid, a degree of cross-linking is 5% to 40% based on a totalnumber of constituent cross-linking group, and a cross-linking extent is0.6% to 15% based on a total number of constituent disaccharide unit ofhyaluronic acid, and a pharmaceutically acceptable carrier.[6] The injection solution as described in any one of [1] to [5],wherein a concentration of a solution of said cross-linked hyaluronicacid derivative is 0.5% by weight to 3.0% by weight based on the totalweight of the solution.[7] The injection solution as described in any one of [1] to [6],wherein a residual ratio in synovial fluid of the cross-linkedhyaluronic acid derivative on 3 days after intra-articularadministration of the solution is not less than 15% compared with thecross-linked hyaluronic acid derivative administered when theconcentration of the solution of the cross-linked hyaluronic acidderivative is about 1% by weight.[8] The injection solution as described in any one of [1] to [6],wherein said analgesic effect lasts or continues for 2 weeks or moreafter administration of the solution at the site of administration.[9] The injection solution as described in [1], comprising thecross-linked hyaluronic acid derivative in the form of unit dosage,wherein the unit dosage comprises 0.3 mg to 1.2 mg per kg of thecross-linked hyaluronic acid derivative on one administration.[10] The injection solution as described in [1], wherein thecross-linked hyaluronic acid derivative has the followingcharacteristics;

a weight average molecular weight of hyaluronic acid is from 500,000 to2,500,000,

a cross-linking group is a residue of cinnamic acid or cinnamic acidderivatives,

a spacer is a residue of aminoalkyl alcohol,

a degree of substitution of a cross-linking group is from 10% to 25%,and

a degree of cross-linking in the cross-linked hyaluronic acid derivativeis from 10% to 30%, and wherein a concentration of the solution of thecross-linked hyaluronic acid derivative is from 0.7% by weight to 2.0%by weight.

[11] A pharmaceutical composition for intra-articular administration fortreating arthritic disorder comprising a cross-linked hyaluronic acidderivative wherein part of carboxyl groups of hyaluronic acid arecross-linked each other with cross-linking group to form crosslinks viaamide bonds and a pharmaceutically acceptable carrier, and having asustainable analgesic effect.[12] The composition as described in [11], wherein a hyaluronic acid iscross-linked to form the cross-linked hyaluronic acid derivative by aphotodimerization reaction or by a photopolymerization reaction withirradiation of light of cross-linking groups having amino group andwherein a carboxyl group of the hyaluronic acid is bound to the aminogroup of the cross-linking group.[13] The composition as described in [11] or [12], wherein the carboxylgroups of the hyaluronic acid are cross-linked with the cross-linkinggroup to an extent of 0.6% to 15% of cross-linking extent based on atotal number of constituent disaccharide unit of hyaluronic acid.[14] The composition as described in any one of [11] to [13], whereinsaid analgesic effect lasts or continues for 2 weeks or more afteradministration of the composition at the site of administration.[15] The composition as described in any one of [11] to [14], wherein aresidual ratio in synovial fluid of the cross-linked hyaluronic acidderivative on 3 days after intra-articular administration of thecomposition is not less than 15% compared with the cross-linkedhyaluronic acid derivative administered when a concentration of asolution of the cross-linked hyaluronic acid derivative is about 1% byweight.[16] An analgesic composition for alleviating joint pain caused byarthritic disorders comprising a cross-linked hyaluronic acid derivativewherein part of functional groups of a hyaluronic acid are cross-linkedwith a cross-linking group to an extent of 0.6% to 15% of cross-linkingextent based on a total number of constituent disaccharide unit ofhyaluronic acid as an active ingredient in such an amount that shows along-lasting analgesic effect or a long-continued analgesic effect, anda pharmaceutically acceptable carrier.[17] A composition for suppressing a cartilage degeneration caused byarthritic disorders comprising a cross-linked hyaluronic acid derivativewherein part of functional groups of a hyaluronic acid are cross-linkedwith a cross-linking group to an extent of 0.6% to 15% of cross-linkingextent based on a total number of constituent disaccharide unit ofhyaluronic acid as an active ingredient in such an amount that shows along-lasting suppressing effect or a long continued suppressing effect,and a pharmaceutically acceptable carrier.[18] A composition for suppressing a inflammation of synovium caused byarthritic disorders comprising a cross-linked hyaluronic acid derivativewherein part of functional groups of a hyaluronic acid are cross-linkedwith a cross-linking group to an extent of 0.6% to 15% of cross-linkingextent based on a total number of constituent disaccharide unit ofhyaluronic acid as an active ingredient in such an amount that shows along-lasting suppressing effect or a long continued suppressing effect,and a pharmaceutically acceptable carrier.[19] The composition as described in any one of [16] to [18], whereinsaid arthritic disorder is osteoarthritis.[20] The composition as described in [19], wherein said arthriticdisorder is traumatic arthritic disorder.[21] A kit comprising a syringe filled with an injection solution or acomposition as described in any one of [1] to [20].

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or the application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication color drawing(s) will be provided by the Patent andTrademark Office upon request and payment of the necessary fee.

FIG. 1 is a graph showing rabbit Anterior Cruciate Ligament (ACL)transection study: gross morphological assessment wherein number ofanimals=12 per group; *P<0.05, **P<0.01 vs. PBS.

FIG. 2A-2D is a histological finding (Safranin O/Fast green) of femoralcondyles on ACL transection study in rabbits.

FIG. 2A is a typical specimen from the phosphate buffer saline (PBS)group.

FIG. 2B is a typical specimen from 1% sodium hyaluronate solution (1%HA-Na) group.

FIG. 2C is a typical specimen from 1 injection Cross-linked hyaluronategel (HA-Gel) group.

FIG. 2D is a typical specimen from 2 injections HA-Gel group.

FIG. 3A-3C is a graph showing analgesic effects of HA-Gel onbradykinin-induced arthritic pain model in rats. Pain scores of PBS,Cross-linked hyaluronate gel (HA-Gel) or 1% HA-Na treated groups,wherein values are shown as mean±S.D. N=12 for each group; *P<0.05,**P<0.01 vs. PBS.

FIG. 3A is a graph showing analgesic effects of HA-Gel administeredintra-articularly 1 week before bradykinin injection.

FIG. 3B is a graph showing analgesic effects of HA-Gel administeredintra-articularly 2 week before bradykinin injection.

FIG. 3C is a graph showing analgesic effects of HA-Gel administeredintra-articularly 4 week before bradykinin injection.

FIG. 4 is a graph showing silver nitrate-induced arthritic pain model:weight loading rate of PBS, HA-Gel or 1% HA-Na treated groups whereinvalues are shown as mean±S.D. N=10 for each group; *P<0.05, **P<0.01 vs.PBS.

FIG. 5 is a graph showing silver nitrate-induced arthritic pain model:pain score of PBS, HA-Gel or 1% HA-Na treated groups wherein values areshown as mean±S.D. N=10 for each group.

FIG. 6 is a graph showing monosodium urate-induced arthritic joint painmodel: changes in abnormal gait score. Physiological saline,Cross-linked hyaluronate gel (HA-Gel) and 1% HA-Na were administeredintra-articularly 0.5 hour before MSU injection. Values are shown asmean±S.D. N=12 for each group.

FIG. 7 is a graph showing monosodium urate-induced arthritic joint painmodel: changes in abnormal gait score. Physiological saline,Cross-linked hyaluronate gel (HA-Gel) and 1% HA-Na were administeredintra-articularly 72 hours before MSU injection. Values are shown asmean±S.D. N=12 for each group.

FIG. 8 is a graph showing residual ratio of HA-Gel in synovial fluid ofthe knees. Values are shown as mean±S.D. N=5 (10 joints) for each groupat each time point; **P<0.01 vs. int-HAD.

FIG. 9 is a graph showing residual ratio of HA-Gel in synovium of theknees. Values are shown as mean±S.D. N=5 (10 joints) for each group ateach time point; *P<0.05, **P<0.01 vs. int-HAD.

FIGS. 10 and 11 are graphs showing papain-induced arthritis model: grossmorphological assessment of cross-linked hyaluronate gels and PBS.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described below in details by wayof detailed embodiments and methods. These detailed description areintended only to be examples of the inventions disclosed and claimedherein and are in no way intended to limit the scope of the inventionwhich is particularly pointed out and distinctly claimed in the numberedclaims appended hereto.

In this specification, the “osteoarthritis”, “hyaluronic acid” and“sodium hyaluronate” are referred to as “OA”, “HA” and “HA-Na”,respectively, and a compound which may be used as a spacer is referredto as “a spacer compound”.

A cross-linked-hyaluronic acid derivative or a cross-linked hyaluronicacid compound to be used in the present invention (hereinafter referredto a cross-linked HA derivative) has a cross-linked structure formed byintra-molecular cross-linking or inter-molecular cross-linking viacross-linking groups which are bonded covalently to HA. The cross-linkedstructure makes the cross-linked HA derivative a three-dimensionalnetworking structure, thereby a solution dissolving the cross-linked HAderivative to an aqueous medium has a physical property of viscoelastichydrogel. The solution has higher viscosity than a HA solution in thesame concentration condition.

The cross-linked HA derivative may be either in a free form of notforming a salt or a pharmaceutically acceptable salt. For example thepharmaceutically acceptable salt of a cross-linked HA derivativeincludes a sodium salt, a potassium salt, a magnesium salt, a calciumsalt and the like.

A hyaluronic acid to be used in the present invention is notparticularly limited in so far as it's a glycosaminoglycan whichconsists of a disaccharide unit consisting of N-acetyl-D-glucosamine andD-glucuronic acid bound through a β1,3 bond as the basic core structureand is constructed by repeating a β1,4 bond of the disaccharide unit,namely a generally used hyaluronic acid (HA).

The HA to be used may be either in a free form of not forming a salt ora pharmaceutically acceptable salt. The pharmaceutically acceptable saltof HA includes salts with alkali metal ions such as a sodium salt, apotassium salt, salts with alkaline earth metal ions such as a magnesiumsalt, and a calcium salt, salt with inorganic base such as ammoniumsalt, and salt with organic base such as diethanolamine, cyclohexylamineand amino acid. The HA salt is preferably a salt with alkali metal ion,particularly a salt with a sodium ion, because of its high affinity forthe living body.

The HA to be used may be derived from natural products by extractingfrom partial materials of living organisms (such as cockscomb, umbilicalcord, cartilage, skin, etc). And it also may be chemically synthesizedor may be produced in microorganisms such as yeast by geneticengineering. Especially it is preferably used HA which has high purityand does not substantially include unacceptable impurities on drug.

The weight average molecular weight of HA is not particularly limited,but from 10,000 to 5,000,000 can be exemplified. Preferably from 200,000to 3,000,000, and more preferably from 500,000 to 2,500,000 can beexemplified.

A cross-linking form to be used in the present invention is preferably across-linking form making use of a covalent bond because of good bindingstability.

A cross-linking group to be used in the present invention is preferablyexemplified a photoreactive cross-linking group (i.e., cross-linkinggroup having a photoreactive residue) which may be selected any residueof compounds capable of undergoing a photodimerization reaction or aphotopolymerization reaction by irradiation with light (ultravioletrays). For example, as said residues of the compounds, there areresidues of cinnamic acid, substituted cinnamic acids, acrylic acid,maleic acid, fumaric acid, sorbic acid, coumarin, thymine or the like.Among these compounds, preferred are those compounds having a vinylenegroup capable of forming a cyclobutane ring by light irradiation, andcinnamic acid or substituted cinnamic acids are more preferred from thepoint of view of photoreactivity and safety for a living body.

The substituted cinnamic acids may be exemplified by cinnamic acidderivatives and the like in which one or two hydrogen atoms at anypositions of the benzene ring of cinnamic acid are substituted by alower alkyl group having 1 to 8 carbon atoms (e.g., methyl, ethyl,propyl, isopropyl, butyl, tert-butyl and the like), a lower alkoxylgroup having 1 to 8 carbon atoms (e.g., methoxy, ethoxy, propoxy,isopropoxy, butoxy and the like), an amino group, a hydroxyl group andthe like, and preferably exemplified by aminocinnamic acid andp-aminocinnamic acid.

The cross-linked HA derivative to be used in the present invention isnecessary to have appropriate resistance to body's internal metabolicenvironment, such as pH, ionic strength, temperature and the like, whichhelps long-lasting or long-continued effects at the administration site.It is preferred to be an amide bond, whereby the residue of compound tobe a cross-linking group is introduced into a carboxyl group of HA,because amide bond has a better resistance to hydrolysis under acidic oralkaline conditions.

The photoreactive cross-linking group above-mentioned may also beintroduced into a carboxyl group of HA via a residue of compounds havingan amino group which is called a spacer. In addition, using a spacerprovides improvement of a reaction with the cross-linking group and HAand a photo cross-linking reaction.

A compound to be used as a spacer in the present invention is notparticularly limited in so far as it is a compound having at least oneamino group and a functional group capable of binding to a photoreactivecross-linking group, for example, aminoalkyl alcohols, diamines, aminoacids, peptides and the like can be cited preferably. Considering adesirable resistant to metabolic degradation and an appropriateconservation of properties on the cross-linked HA derivative during anintra-articular administration, it is preferably exemplified byaminoalkyl alcohols having from 2 to 18 carbon atoms, and morepreferably aminoalkyl alcohols having 2 to 12 carbon atoms. Especiallypreferably aminopentanol, aminobutanol, aminopropanol and aminoethanolcan be exemplified because of giving a suitable distance between HA anda photoreactive cross-linking group on a cross-linking reaction.

Unless otherwise noted, in this specification, the cross-linking groupmay include a cross-linking group which is introduced into a residue ofa spacer compound, accordingly a photoreactive cross-linking group mayalso include a photoreactive cross-linking group which is introducedinto a residue of a spacer compound. And a cross-linking group whichforms a cross-linked structure is occasionally referred to as across-linked group and a cross-linking group which is not cross-linkedis sometimes referred to as a cross-linkable group.

A method for synthesizing the cross-linked HA derivative of the presentinvention is not particularly limited, in so far as it is a method forbeing possible to bind chemically HA to a compound which can be across-linking group via amide bond, for example, a method using awater-soluble condensing agent such as a water-soluble carbodiimide,e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDCI.HCl), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate,1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide hydrochloride and thelike; a method using an auxiliary condensing agent such as N-hydroxysuccinimide (HOSu) and N-hydroxy benzotriazole (HOBO in addition to theabove condensing agent; a method using a condensing agent such as4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM) and the like; active esterification method; acid anhydridemethod and the like. In addition, when a cross-linking group into whicha spacer is introduced is used as a cross-linking group, it may beeither a method in which a spacer compound is introduced into HA inadvance and then a cross-linking group is introduced into thespacer-linked HA or a method in which a spacer is introduced into across-linking group in advance, and then the spacer-linked cross-linkinggroup is introduced into HA.

Hereinafter, for simplify a representation, a cross-linking group-linkedHA derivative produced by the above described method without building across-linked structure yet is occasionally referred to as anon-cross-linked product or a non-cross-linked compound.

A method for crosslinking a non-cross-linked product is not particularlylimited, in so far as a method to may produce to form a cross-linkedstructure by reacting between cross-linking groups.

For example, in case of the non-cross-linked product using a photocross-linking group, it is preferred to a method by irradiating asolution dissolving the non-cross-linked product homogeneously withlight.

Although HA in itself has good solubility in an aqueous medium, in caseof hyaluronic acid derivative in which a spacer compound is introducedinto to the carboxyl group owned by hyaluronic acid which contribute toit's hydrophilic property, the hydrophilic property decreases as thedegree of substitution increases.

Therefore, the above described method for synthesizing thenon-cross-linked product preferably includes a method which comprisescarrying out an alkali treatment for improving a solubility in anaqueous medium of the non-cross-linked product.

A method of alkali treatment is not particularly limited, in so far asit is a treatment by which a solution becomes alkaline. An alkali agentused may be in the form of either organic or inorganic salts. Inconsideration of treatment in the aqueous solvent, the use of theinorganic alkali salts is preferred. Among these inorganic alkali salts,weak alkali salts such as sodium hydrogen carbonate and sodium carbonateare more suitably used as compared to strong alkali salts such as sodiumhydroxide since such weak alkali salts have a less influence onconversion of cross-linking group-linked hyaluronic acid derivative intolow-molecular compounds or decomposition of the cross-linking group.Here, the alkali treatment may be conducted at a pH value of usually 7.2to 11, preferably 7.5 to 10. The amount of the alkali used and thealkali-treating time may be appropriately controlled depending upon theaimed hydrophilic property. For example, when sodium hydrogen carbonateis used in an amount of 500 mg based on 1 g of hyaluronic acid (i.e., ina molar amount 10 times or more the mole of hyaluronic acid), the alkalitreatment may be conducted for 2 to 3 hours under stirring, therebyobtaining the non-cross-linked compound having a sufficiently enhancedhydrophilic property. A 1.0% by weight solution of the non-cross-linkedderivative obtained by the above method is capable of passing through aporous filter having a pore size of 0.45 μm and a diameter of 25 mm at arate of not less than 2 mL/minute at 24° C. under a pressure of 5.0Kg/cm².

It is preferred that light irradiation (photoirradiation) of anon-cross-linked product is carried out under such conditions that thephotoreactive cross-linking group efficiently causes a photodimerizationor a photopolymerization reaction.

A kind and wavelength of light to be used is not particularly limited,in so far as a light which may be selected from those having awavelength capable of subjecting the photoreactive cross-linking groupto a photoreaction without cleavage of glycoside bond of the hyaluronicacid. For example, when a cross-linking group to be used is a cinnamicacid or a cinnamic acid derivative, an ultraviolet light having awavelength of 200 to 600 nm is preferred. An integration of theirradiated light is appropriately selected depending on a desiredproperty of the resultant, a desired degree of substitution, aconcentration of the non-cross-linked product solution and the like. Asa preferred light source, there may be used an ultraviolet lamp, ahigh-pressure mercury lamp or a metal halide lamp. Preferably, ifnecessary, undesired wavelengths may be removed from the light source,for example, by a cut filter.

The cross-linked HA derivative of the present invention has across-linked structure formed by intra-molecular cross-linking orinter-molecular cross-linking via cross-linking groups which are bondedcovalently to HA. The cross-linked structure makes the cross-linked HAderivative a three-dimensional networking structure, thereby a solutionof the cross-linked HA derivative has a physical property ofviscoelastic hydrogel composed of viscous and elastic. The physicalproperty is affected by contributing factors such as a degree ofsubstitution of a cross-linking group, a degree of cross-linking, aconcentration of a cross-linkable compound on cross-linking reaction andthe like. And therefore it is important to set up these factors at anappropriate range.

These suitable ranges may be decided appropriately depending on adesired property of a resultant. For example, a degree of substitutionof a cross-linking group of the present invention is preferably 3% to50%, more preferably 5% to 30% and much more preferably 10% to 25% basedon a total number of constituent disaccharide unit of HA. A preferredconcentration of a reaction solution on cross-linking reaction may beexemplified 0.5% to 10%, more preferably 0.7% to 2% based on a totalweight of the solution. A degree of cross-linking is exemplifiedpreferably 5% to 40%, more preferably 7% to 35% and much more preferably10% to 30% based on a total number of constituent cross-linking group.

In addition, the degree of substitution (DS) can be calculated on theratio (%) of introduction of a cross-linking group per constituentdisaccharide unit of HA, for example, DS on the non-cross-linkedcompound having one cross-linking group per constituent disaccharideunit or one cross-linking group per constituent 200 saccharide units is100% or 1%, respectively.

The degree of cross-linking can be calculated on the ratio (%) ofcross-linked groups to the introduced cross-linking groups. For example,in the case of the HA derivatives having 100 cross-linking groups if 20cross-linking groups (monomers) react into 10 dimmers, the degree ofcross-linking is 20%.

It is commonly agreed that it is difficult to get a hydro-gel solutionhaving moderate fluidity if a cross-linked hyaluronic acid derivative tobe a solute are excessive amount with compared to medium (solvent) andthere are excessive bridge formation in a cross-linked hyaluronic acidderivative.

The cross-linked HA derivative of the present invention needs a propertyof moderate fluidity for intra-articular administration by aneedle-tipped syringe. Meanwhile it needs also an appropriate resistanceto body's internal metabolic environment and a suitable retentioncapability at administration site. Therefore, it is not preferred to bemuch low DS or degree of cross-linking. Consequently, a balance ofviscosity and elasticity is important.

The solution of a cross-linked HA derivative designed according to theabove mentioned conditions can be an injectable solution capable passingthrough a needle having from 18 gauge to 25 gauge and can be used aninjection solution for intra-articular administration.

For example, from the standpoint of a cross-linking extent which is aproduct of DS and a degree of cross-linking and is expressed as in termsof molar ratio (%) of dimmers of cross-linking group per constituentdisaccharide unit of HA, the cross-linked HA derivative of the presentinvention is preferred to have a cross-linking extent in the range from0.6% to 15%, more preferably in the range from 1.0% to 7.5% based on atotal number of constituent disaccharide unit of HA.

As shown in the examples which are described below, the cross-linked HAderivative of the present invention showed a more significant analgesiceffect by a single injection, not by a series of 3 to 5 injections forone treatment period. And it also showed keeping an analgesic effect fora long term than a solution of HA alone, and showed an effective andlong-lasting or long-continued analgesic effect on both acute pain andchronic pain. Moreover, it also provides an anti-inflammatory effect onsynovium and a cartilage protective effect for a long term by a singletime injection.

According to the present invention, it is possible to provide aninjection solution and a pharmaceutical composition for intra-articularadministration for treating arthritic disorders including OA, traumaticarthritis, inflammatory arthritic disorder, degenerative arthriticdisorder and the like. The injection solution comprises the cross-linkedhyaluronic acid derivative of the present invention as an activeingredient and a pharmaceutically acceptable carrier, and similarly thepharmaceutical composition comprises the cross-linked hyaluronic acidderivative of the present invention as an active ingredient and apharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier is exemplified an aqueous mediumas which is used for a solution of the cross-linked HA derivative of thepresent invention. For example there are water for injection,physiological saline, and phosphate buffered saline. Also it may beacceptable an additive to be usually used for the injection such as anpH adjuster and tonicity agent unless the injection solution and thepharmaceutical composition of the present invention lose a desiredtherapeutic effect and produce side-effects. For example there aresodium dihydrogen phosphate, disodium hydrogen phosphate, and sodiumchloride.

The injection solution and the pharmaceutical composition are effectivefully by a fewer number of doses for a duration of therapy than thenumber of doses of commercially available IA-HA products (i.e., a seriesof 3 to 5 times injections for a duration of therapy).

And also it is possible to provide a kit comprising a syringe filledwith a solution of the cross-linked HA derivative of the presentinvention, if necessary, which may be equipped with a plunger, a plungerrod and the like.

A cross-linked HA derivative produced according to the above mentionedmethod and conditions can be used for the injection solution and thepharmaceutical composition. Preferable cross-linked HA used for theinjection solution or the pharmaceutical composition has the followingparameters as for cross-link;

a cross-linking extent of from 0.6% to 15% and a degree of substitutionof from 3% to 50%,

a cross-linking extent of from 0.6% to 15% and a degree of cross-linkingof from 5% to 40%,

a degree of substitution of from 3% to 50% and a degree of cross-linkingof from 5% to 40%,

a cross-linking extent of from 0.6% to 15%, a degree of substitution offrom 3% to 50%, and a degree of cross-linking of from 5% to 40%, or

a cross-linking extent of from 1% to 7.5%, a degree of substitution offrom 10% to 25%, and a degree of cross-linking of from 10% to 30%.

A disease to be treated by the pharmaceutical agent of the presentinvention is not particularly limited, and it is possible to be used asa therapeutic agent for alleviating joint pain caused by arthriticdisorders and suppressing an inflammation of synovium caused byarthritic disorders, suppressing a cartilage degeneration caused byarthritic disorders and improving a range of motion on joint. Inaddition, it is possible to use the same not only for treatment but alsofor prevention of the above-mentioned diseases.

A dose of the injection solution or the pharmaceutical composition isnot particularly limited, because it is an item which should beindividually decided according to specific symptoms, age, body weightand the like of the subject to be treated.

Preferably the normal dose range of 15 mg to 60 mg per adult patient(50-70 kg) on one administration based on a HA derivative can beexemplified, for more information the range of 0.3 mg to 1.2 mg per kgcan be exemplified. And preferably the concentration of the injectionsolution can be exemplified from 0.5% to 3.0%, more preferably from 0.7%to 2.0% (as a cross-linking group-linked HA derivative) based on thetotal weight of the solution. The usage of the injection solution maypreferably be exemplified single injection or two injections for oneseries of treatment. An injectable form of the pharmaceuticalcomposition is the same.

One of the preferable embodiments of the injection solution and theinjectable form of the pharmaceutical composition of the presentinvention can be exemplified below.

The properties of the cross-linked HA derivative are the following:

the weight average molecular weight of HA: 500,000 to 2,500,000,

the compound of cross-linking group: cinnamic acid or cinnamic acidderivatives,

the spacer compound: aminoalkyl alcohol, more preferably aminopentanol,aininobutanol, aminopropanol or aminoethanol,

the DS of a cross-linking group: from 10% to 25%, and

the degree of cross-linking: from 10% to 30%.

Medium: saline, phosphate buffered saline or water for injection

Concentration of the solution of cross-linked HA derivative: from 0.7%to 2.0%

Properties of the solution: having higher viscoelastic character andhigher tread-formability than a solution of HA in the same concentrationcondition, and being capable of passing through a needle having from 18to 25 gauge when extruded from the injection needle at 24° C. at a rateof 0.2 ml/second.

Moreover, the cross-linking extent can be preferably exemplified from1.0% to 7.5%. And it has a moderate flowability, for example, the 1% byweight (as a cross-linking group-linked HA) of the cross-linked HAderivative gel is possible to form a continuous thread having a lengthof not less than 3 cm which is formed without break from a tip open endof an 23-gauge injection needle when extruded from the injection needleat 24° C. at a rate of 0.2 ml/second in the direction of 45° downwardfrom a horizontal direction.

In the present invention, a hydrogel state solution comprisingcross-linked HA derivatives is mentioned in, for example, U.S. Pat. No.6,602,859 the contents of which are incorporated herein by reference.

EXAMPLES

The present invention is described below more specifically based onExamples. However, there is no intention to limit the technical scope ofthe present invention by this.

Synthetic Example

400 mg of sodium hyaluronate having a weight-average molecular weight of900,000 (manufactured by SEIKAGAKU CORPORATION) was mixed with a mixedsolution containing water and dioxane under stirring. To the resultantsolution was sequentially mixed 34 mg of N-hydroxy succinimide (HOSu)/1ml of water (0.6 equivalent/HA disaccharide unit (mol/mol)); 29 mg of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDCI.HCl)/1 ml of water (0.3 equivalent/HA disaccharide unit(mol/mol)); 51 mg of 4-(6-aminohexanamide)ethyl cinnamatehydrochloride/1 ml of water (0.3 equivalent/HA disaccharide unit(mol/mol)), at room temperature. The resultant mixture was stirred for 3hours. The obtained mixture was further mixed with 200 mg of sodiumhydrogen carbonate/3 ml of water, stirred for 2 hours, and then mixedwith 400 mg of sodium chloride. Ethanol was charged into the resultantreaction solution to precipitate solid. The obtained solid wassuccessively washed with 80% (vol./vol.) ethanol and ethanol, and thendried overnight at room temperature, thereby obtaining 360 mg of a whitesolid (4-(6-aminohexanamide)ethyl cinnamate-bound hyaluronic acid:“4-(6-aminohexanamide)ethyl cinnamate-bound hyaluronic acid” ishereinafter referred to merely int-HAD 1.

Further, the same procedure as above was conducted except that theequivalent amounts of N-hydroxy succinimide (HOSu), EDCI.HCl andcinnamate derivatives were changed as described hereinbelow, therebyobtaining int-HAD having different introduction percentages (degree ofsubstitution) from each other.

(int-HAD 2) HOSu, EDCl.HCl and 3-aminopropyl cinnamate hydrochloridewere respectively 0.2, 0.10 and 0.10 mol/mol HA disaccharide unit.

(int-HAD 3) HOSu, EDCl.HCl and 4-aminobutyl cinnamate hydrochloride wererespectively 1.0, 0.5 and 0.5 mol/mol HA disaccharide unit.

(int-HAD 4) HOSu, EDCl.HCl and 5-aminopentyl cinnamate hydrochloridewere respectively 1.4, 0.7 and 0.7 mol/mol HA disaccharide unit.

(int-HAD 5) HOSu, EDCl.HCl and 8-aminooctyl cinnamate hydrochloride wererespectively 0.6, 0.3 and 0.3 mol/mol HA disaccharide unit.

(int-HAD 6) HOSu, EDCl.HCl and 3-aminopropyl cinnamate hydrochloridewere respectively 0.2, 0.10 and 0.10 mol/mol HA disaccharide unit. Theint-HAD 6 was prepared by the same method of int-HAD 2 preparation,except that the degree of 3-aminopropyl cinnamate was lower than that ofint-HAD 2.

Then, the cinnamic acid derivative-introduced hyaluronic acids obtainedfrom above (int-HADs 1-6) are respectively dissolved in a 5 mMphosphate-buffered physiological saline such that the concentration ofthe obtained solution became 1.0% by weight calculated as hyaluronicacid.

And the obtained solutions were respectively irradiated with ultravioletrays for 20-40 minutes. These solutions were changed to gel form(hydrogel) by ultraviolet irradiation. The obtained gel is hereinafterreferred to Crosslinked hyaluronate gel.

“Cross-linked hyaluronate gel” in the following experimental studies(herein occasionally referred to HA-Gel) was the gel which wasirradiated with ultraviolet ray to 1.0% solution of int-HAD prepared bythe above synthetic example. “1% sodium hyaluronate solution (1% HA-Na)”was used SUPARTZ® manufactured by SEIKAGAKU CORPORATION in the followingexperimental studies (hereinafter occasionally referred to HA-Na).

Both Cross-linked hyaluronate gel and 1% sodium hyaluronate solution (1%HA-Na) were used hyaluronic acid derived from chicken/rooster comb.

Example 1 Effects of Intra-articular Injection of Cross-LinkedHyaluronate Gel (HA-Gel) on the Anterior Cruciate Ligament (ACL)Transection Induced Arthritis in Rabbits Objective

The rabbit ACL transection model used in this study has been accepted asan arthritis model, which produces cartilage degradation similar to OAin humans. Therefore, this model has been frequently used for evaluationof hyaluronan preparations (SUPARTZ®, HYALGAN®, HEALON®, SYNVISC®).

The objective of this study was to examine the efficacy of HA-Gel in theACL transection model.

Methods

Experimental osteoarthritis (OA) was induced in 48 male rabbits bytransecting the unilateral ACL. Four weeks after the ACL transection,HA-Gel was administered once or twice (at an interval of 2 weeks) intothe joint cavity of the left hind knee at a dose of 0.05 mL/kg/joint.Its efficacy was compared to that of repeated administration of PBS or1% sodium hyaluronate solution (1% HA-Na) once a week for 5 weeks. Allanimals were sacrificed 9 weeks after ACL transection. The left kneejoints were then removed and evaluated by morphological assessment ofcartilage degeneration, a volume of synovial fluid and protein content,a number of infiltrated cells and a glycosaminoglycans content insynovial fluid as indicators of synovitis, and safranin O stain offemoral condyles as histopathological examination of the cartilage andsynovium.

The injection volume was set the same among all test substances.

Results

In morphological assessment, each animal was assessed for changes in twosites (femoral condyles and tibial plateaus) and total of 24 sites ineach group were graded according to the following criteria:

Grade 1: Intact surface (No staining by Indian ink)

Grade 2: Minimal fibrillation (Surface retains the ink as elongatedspecks)

Grade 3: Overt fibrillation

Grade 4: Erosion (Loss of cartilage exposing the underlying bone)

4a: 0 mm<Erosion≦2 mm in length

4b: 2 mm<Erosion≦5 mm in length

4c: 5 mm<Erosion in length

4d: 5 mm×2 mm<Erosion (mm in length×width)

In the morphological assessment, cartilage degeneration was less in theHA-Gel groups compared to the 1% HA-Na (FIG. 1). In fact, in both theone- and two-injections HA-Gel groups, the degeneration of the articularcartilage was significantly less compared with control treated with5-weekly injections of PBS. Moreover, considering the number ofinjections, the HA-Gel clearly showed to decrease the degeneration ofthe articular cartilage compared with 1% HA-Na. In contrast, 1% HA-Naadministration decreased the cartilage degeneration compared with PBS,but the efficacy was not significant. The severity comparison among thetest substances were in the following order: PBS>1% HA-Na>1 injection ofHA-Gel≧2 injections of HA-Gel. Efficacy of HA-Gel for suppression ofcartilage degeneration was also demonstrated by a reduction of theincrease in chondroitin 6-sulfate (CS-6S) in the synovial fluid. Inaddition, HA-Gel appeared to improve the symptoms of synovitis, asjudged from the reduction in increase of synovial fluid, protein andchondroitin 4-sulfate (CS-4S) contents.

In addition, that is reported that CS-6S in the synovial fluid isoriginated from cartilage and CS-4S in the synovial fluid is releasedthrough the blood vessels by inflammatory of synovial membrane. In otherwords, the reduction of the increase of CS-6S indicated to prevent thecartilage degeneration and the reduction of the increase of CS-4Sindicated to prevent the inflammatory of synovial membrane.

In FIG. 2A-2D, (A) is a typical specimen from the PBS group, (B) is atypical specimen from 1% HA-Na group, (C) is a typical specimen from 1injection HA-Gel group, and (D) is a typical specimen from 2 injectionsHA-Gel group (original magnification×40).

The Safranin 0 stain displays glycosaminoglycans (GAGs) content withinthe cartilage (GAGs: red, Bone & collagen fibers: green).

The following observations were carried out and scored: area ofcartilage matrix unstained/decreased staining for Safranin O, fissureformation in cartilage matrix, fibrillation of cartilage matrix, defectof cartilage, increase in number of chondrocytes, decrease in number ofchondrocytes, remodeling of sub-cartilaginous bone and blood vesselinvasion in cartilage matrix. The severity comparison among the testsubstances were in the following order: PBS>1% HA-Na>1 injection ofHA-Gel>2 injections of HA-Gel.

Overall, since cartilage degeneration is milder when synovitis is notsevere, these changes induced by HA-Gel may interact beneficially torelieve the progression of pathological changes. Histopathologicalfindings of articular cartilage supported the macroscopic assessment(FIG. 2A-2D). In the histopathological examination of synovium,cuboidal/stratified synovial epithelium, subepithelial cellularinfiltration, subepithelial fibrosis/edema, subepithelial hemorrhage andsubepithelial calcium deposition were observed in all the experimentalgroups. These changes were less severe in HA-Gel groups compared tothose in the PBS group.

Conclusion

These data show that in a rabbit ACL transection model of OA, theadministration of HA-Gel once or twice (at an interval of 2 weeks)suppressed both synovitis and cartilage degeneration.

Example 2 Effects of Intra-Articular Injection of Cross-LinkedHyaluronate Gel (HA-Gel) on a Bradykinin-Induced Arthritic Pain in RatsObjective

The arthritic pain model used in this study is a local pain modelproduced by injecting bradykinin (an endogenous hyperalgesic substance)with PGEs (a pain enhancer) into the joint cavity of rats. This modelhas been used to assess the analgesic effects of hyaluronan preparationsbased on the behavioral manifestations of joint pain in gait such as“lifting the foot”, “claudication” and “walking on three legs”. It hasbeen reported that the analgesic effect assessed by this model wasassociated with the concentration of hyaluronan in synovial tissue.

The objective of this study was to examine the long-lasting analgesiceffect of HA-Gel on arthritic pain induced by bradykinin in ratscompared to 1% HA-Na and PBS.

Methods

The test substance was administered into the knee joint cavity of theleft hind leg of female rats at a dose of 0.05 mL/joint. On weeks 1, 2and 4 after administration, the mixture of bradykinin solutions and PGE2were injected into the same joint to induce arthritic pain. 1% HA-Na wasonly evaluated at 1 week, since this treatment typically requiresreinjection once a week. Under blind conditions, the walking of theanimal was observed for about 2 minutes after injection of bradykininsolutions and the severity of pain was scored on a 5-point scale (Table1).

TABLE 1 Bradykinin-induced arthritic pain model: Criteria for assigningpain scores Score BEHAVIORAL MANIFESTATION 0 Normal or claudication for≦5 sec 1 Claudication for 6-30 sec 2 Showing one of the following twomanifestations: Claudication for ≧31 sec Lifting the foot for ≦5 sec 3Showing one of the following two manifestations: Lifting foot followedby claudication Walking on three legs for ≦5 sec 4 Walking on three legsfor ≧6 sec

Results

HA-Gel significantly suppressed the bradykinin-induced pain responsecompared with control group at the time points 1, 2 and 4 weeks postadministration (FIGS. 3A-3C). The analgesic effect by HA-Gel was moreremarkable than that by 1% HA-Na at 1 week.

Conclusion

It was demonstrated that HA-Gel administered into the joint cavity wasmore effective against bradykinin-induced arthritic pain than PBS and 1%HA-Na tested as the positive control. As compared to controls, HA-Gelprovided a sustained analgesic effect that lasted for at least 4 weeks.

Example 3 Effects of Intra-articular Injection of Cross-LinkedHyaluronate Gel (HA-Gel) on the Silver Nitrate-Induced Arthritic Pain inRats Objective

The objective of this study was to examine the pain-relieving effect ofHA-Gel on arthritic pain induced by silver nitrate in rats³. This modelhas been commonly used for the evaluation of the analgesic effect ofnumerous non-steroidal anti-inflammatory drugs (NSAIDs).

Methods

A 1% silver nitrate aqueous solution was injected into the knee jointcavity of the left hind leg of 42 male rats at a dose of 0.05 mL/joint.At 24 hours after injection, animals were allocated to groups accordingto the pain score while walking and the weight loading on the inflamedpaw. The severity of pain was scored on the following 4-point scale; 0point for normal or nearly normal, 1 score for mild claudication, 2score for severe claudication and 3 score for walking on three legs. Theweight loading was measured by using a weighting activity analgesiameter (manufactured by Tokken Inc.). HA-Gel, 1% HA-Na or PBS wasadministered at a dose of 0.05 mL/joint. On days 1, 2 and 3 afteradministration, the weight loading on the inflamed paw and pain scorewere assessed in the above same way. Items evaluated were the weightloading rate (%) (=loading on inflamed paw (g)/body weight (g)×100) andpain score while walking

Results

At each time point on days 1 to 3 after administration of the testsubstance, the weight loading rate on the inflamed paw was significantlyhigher in animals given HA-Gel compared to those given PBS or 1% HA-Na(FIG. 4). In addition, the pain score of HA-Gel was lower than PBS or 1%HA-Na at each time point measured (FIG. 5).

Conclusion

These data demonstrate that HA-Gel administered into the joint cavitywas more effective for inflammatory joint pain than PBS and 1% HA-Na.

Example 4 Analgesic Effect of Cross-Linked Hyaluronate Gel (HA-Gel) onMonosodium Urate-Induced Arthritic Joint Pain in Dogs Objective

The deposit of micro-crystals of monosodium urate (MSU) in synovialfluid promotes acute inflammatory joint pain in humans. The experimentaljoint pain model induced by an intra-articular injection of MSU crystalshas been widely used to evaluate the analgesic efficacy of sodiumhyaluronate and non-steroidal anti-inflammatory drugs.

The objective of this study was to examine the analgesic effect ofHA-Gel on joint pain induced by MSU in dogs.

Methods

HA-Gel, physiological saline or 1% HA-Na was administered to femalebeagle dogs by an intra-articular injection into the knee joint cavityof the left hind limb at a dose of 0.3 mL/kg/joint. Monosodium urate(MSU), a substance which induces inflammation and severe pain, was theninjected at the same joint 0.5 or 72 hours after the test materialtreatment. At 2, 3, 4, 5, 6 and 8 hours after the injection of MSU, gaitcondition was scored and weight-bearing rates of the left hind limb werecalculated as the joint pain indicators. The area under the time curve(AUC) was calculated from both these indicators.

Gait was observed and the condition of the gait was scored (indicated asabnormal gait score) according to the following criteria; 0: Unchanged(Normal gait), 1: Mild (Stands normally but gait is unnatural), 2:Moderate (Stands on 4 limbs but often raises left hind limb (injectionsite)), 3: Severe (Touches only the tip of the left hind limb to theground when walking) and 4: Very severe (Cannot put any weight on lefthind limb; walking on 3 limbs). In addition, three scales were used forthe weight-bearing measurement: scale A was a digital platform scale(DP-6100GP; Yamato Scale Co., Ltd); and scales B and C were load-celldigital platform scales (DP-6000; Yamato Scale Co., Ltd.). The frontlimbs were placed on scale A, the right hind limb on scale B, and theleft hind limb on scale C. The values from all three scales wererecorded to the nearest 0.1 kg. The weight-bearing rate was calculatedby using the following formula.Weight-bearing rate=100×Mean C/(Mean A+Mean B+Mean C)

Results

In experiments where MSU was injected 0.5 hour after the test materialtreatment, the mean AUCs of abnormal gait score were 0.0, 28.0 and 13.5in HA-Gel, physiological saline and 1% HA-Na, respectively (FIG. 6,Table 2). HA-Gel exhibited a complete analgesic effect and no painfulwalking was observed in all of the treated animals. 1% HA-Na alsoexhibited a significant analgesic effect, as compared to physiologicalsaline, but the effect was significantly lower than that of HA-Gel.

In experiments where MSU was injected 72 hours after the test materialtreatment, the AUCs of abnormal gait score were 1.5, 27.6 and 27.1 inHA-Gel, physiological saline and 1% HA-Na, respectively (FIG. 7, Table3). HA-Gel still exhibited a significant analgesic effect, whereas theeffect of 1% HA-Na on the knee joint pain was almost completelydiminished.

In both the experiments, changes in the weight-bearing rates of the lefthind limb were consistent with those of the abnormal gait scores.

TABLE 2 Monosodium urate-induced arthritic joint pain model: AUC ofabnormal gait score. Physiological saline, HA-Gel and 1% HA-Na wereadministered intra-articularly 0.5 hours before MSU injection. Number ofAUC of abnormal Group animals gait score Physiological saline (0.3 12  28.0 ± 0.0 mL/kg) Cross-linked 12   0.0** ± 0.0 hyaluronate gel (HA-Gel) (0.3 mL/kg) 1% HA-Na (0.3 mL/kg) 12 13.5**^(,##) ± 3.2 **p < 0.01:Significant difference from Physiological saline (Tukey-Kramer multiplecomparison test) ^(##)p < 0.01: Significant difference from Cross-linkedhyaluronate gel (Tukey-Kramer multiple comparison test)

TABLE 3 Monosodium urate-induced arthritic joint pain model: AUC ofabnormal gait score. Physiological saline, HA-Gel and 1% HA-Na wereadministered intra-articularly 72 hours before MSU injection. Number ofAUC of abnormal Group animals gait score Physiological saline (0.3 12 27.6 ± 1.1 mL/kg) Cross-linked hyaluronate gel (HA- 12 1.5** ± 4.5 Gel)(0.3 mL/kg) 1% HA-Na (0.3 mL/kg) 12 27.1^(##) ± 2.1  **p < 0.01:Significant difference from Physiological saline (Tukey-Kramer multiplecomparison test) ^(##)p < 0.01: Significant difference from Cross-linkedhyaluronate gel (Tukey-Kramer multiple comparison test)

Conclusion

It was confirmed that the single intra-articular administration ofHA-Gel suppressed the MSU-induced arthritic knee joint pain. Compared to1%HA-Na, the analgesic effect of HA-Gel was superior and more prolonged.

Example 5 Residual Ratio of Cross-Linked Hyaluronate Gel (HA-Gel) inJoint Cavity and Synovium of the Knee in Rabbits Objective

The objective of the study was to investigate the local retention ofintra-articularly injected HA-Gel in rabbits.

Methods

HA-Gel and its non-cross-linked intermediate (int-HAD) were administeredinto the both right and left knee joint cavity in male rabbits at a doseof 0.05 mL/kg/joint (the concentrations of HA-Gel and int-HAD: 1%).Animals were sacrificed on days 1, 3, 5, 7, 14 and 28 afteradministration, and synovial fluid and synovium were collected. Aremaining cross-linking agent of Cross-linked hyaluronate gel,trans-cinnamic acid (tCA), was quantified by high performance liquidchromatography (HPLC) to calculate the residual ratios of HA-Gel andint-HAD.

Results

Residual percentage was calculated from the measured value by HPLC.

About the metabolic disposition of the hyaluronate with extraneousadministration to the joint cavity it is known commonly that thehyaluronate migrates gradually from the synovial fluid to the synovium.

Most of the HA-Gel disappeared from the synovial fluid within 7 days,however, it remained in the synovium for up to 28 days afteradministration. By comparing the residual ratios of HA-Gel and int-HAD,significantly higher levels of HA-Gel were detected in the synovialfluid on days 1, 3, and 5 after administration (FIG. 8). According toFIG. 8, the HA-Gel remained in the synovial fluid to about 15% of theadministered HA-Gel on 3 days after administration. However, in thesynovium, the ratios of Cross-linked hyaluronate gel remainedsignificantly higher on days 7, 14, and 28 (FIG. 9).

Conclusion

Compared to non-cross-linked hyaluronan, the injected HA-Gel remained insynovial fluid and synovium for a prolonged period of time. On the otherhand, non-cross-linked hyaluronan diffused out of the synovial fluidrapidly, with lower levels retained in the synovium. So, HA-Gel canremain in long-term existence in the joint (i.e., at administrationsite), because it migrates more slowly from the synovial fluid to thesynovium and is more gradually metabolized in comparison with int-HA.The increased retention of HA-Gel may contribute to a superior cartilageprotective effect and a long-lasting analgesic effect.

Example 6 Effects of Intra-articular Injection of Cross-LinkedHyaluronate Gel (HA-Gel) on the Papain-Induced Arthritis in Rabbits

The rabbit papain induced arthritis model used in this study has beenaccepted an osteoarthritis (OA) model produced by injecting papain (acysteine protease present in papaya) into the knee joint cavity ofrabbits.

The objective of this study was to examine the efficacy of HA-Gel in thepapain-induced arthritis model. This study was performed twice.

Rabbits (21-week-old male) were fixed in a supine position underketamine general anesthesia (1 mL/head, i.v.), and a wide area aroundthe knee joint of the left hind leg was shaved with an electric clipper.The injection site of the knee joint was sterilized with 70% ethanol andIsocline® solution.

After that, 0.8% papain solution was administered twice (at an intervalof 3 days) into the joint cavity of the left hind knee at a dose of 500μL/joint.

Total 20 rabbits, 5 rabbits for each group, were used in this study. Thepapain solution was activated by L-Cysteine immediately prior to theinjection.

One week after the second injection of papain, 150 μL of the testsubstance (HA-Gel or phosphate buffered saline (PBS)) was administeredinto the knee joint cavity of the left hind leg once per week for 3weeks.

All animals were sacrificed 1 week after the last administration of thetest substances. The left knee joints were then removed and synovialfluid and synovium were collected from the left knee joint.

The left knee joints were evaluated by morphological examination,histopathological examination of the synovium and articular cartilage,volume of synovial fluid and protein content. As histopathologicalexaminations, paraffin sections were made from formalin-fixed synoviumand stained with hematoxylineosin (HE) and alucian-blue. After the EDTAdecalcification and safranin O staining, the cartilage conditions wereobserved in the femoral condyles and tibial plateaus.

In the morphological assessment, degeneration severity was scored on thesame criteria of Example 1 (FIGS. 10 and 11). Cross-linked hyaluronategels alleviated the cartilage degeneration compared to PBS. However, theefficacy was not significant. It is considered that the number ofanimals is not enough.

In the histopathological examination of cartilage, the degeneration ofcartilage matrix, chondrocyte decrement and decreased staining forsafranin O were alleviated by HA-Gel.

HA-Gel used in this study suppressed the cartilage degeneration comparedto PBS.

Cross-linking extents of cross-linked hyaluronate gels used in thisstudy were respectively 1.72% (FIG. 10) and 2.06% (FIG. 11) and waslower than HA-Gel used in other example studies. It is considered thatthe degree of the cross-linking extent is essential to achievesignificant efficacy on improvement of the arthritic pain and thecartilage degeneration.

INDUSTRIAL APPLICABILITY

According to these above examples, it was demonstrated that theintra-articular injection of HA-Gel once or twice (at an interval of 2weeks) had the same or much higher effectiveness than five injections ofHA-Na for treatment of knee OA. And also it is confirmed that HA-Gelalso provided a therapeutic efficacy in animal models for evaluation ofNSAIDs.

When HA-Gel was injected into the knee joint cavity, most of the HA-Geldisappeared from the synovial fluid within 7 days. However, it remainedin the synovium for up to 28 days after administration. Since it isconsidered that the concentration of hyaluronan in synoviun correlatedwith the analgesic effect, the increased retention of HA-Gel maycontribute to a long-lasting analgesic effect. Moreover, HA-Gel isexpected to be well shock absorber to relieve pain and improve kneefunction in OA, since it has a higher visco-elasticity than HA-Nasolution.

It may be proposed a therapeutic agent for joint diseases which has thefollowing merits; a long-lasting effect by single injection, a decreaseof number of doses for one series of treatment, and a reduction of therisk of infection in an intra-articular administration. Consequentlystresses on affected patients of joint disorders can be lowered.

The invention claimed is:
 1. A solution of a hyaluronic acid derivativein a pharmaceutically acceptable aqueous medium in an amount of 0.1% to2% by weight based upon the total weight of the solution, the hyaluronicacid derivative comprising cross-linkable groups introduced into somefunctional groups of hyaluronic acid, the hyaluronic acid derivativehaving following characteristics: (1) the cross-linkable groups are aresidue of cinnamic acid or a cinnamic acid derivative, and (2) a degreeof substitution of the cross-linkable groups is from 10% to 25%, whereinat least one of the following is present: (A) the residue of cinnamicacid or a cinnamic acid derivative is attached to the functional groupsof the hyaluronic acid by an amide bond, and wherein the functionalgroups are carboxy groups; and (B) the hyaluronic acid derivative has aspacer between the cross-linkable groups and the functional groups ofthe hyaluronic acid; and wherein the hyaluronic acid derivative iscapable of being cross-linked into a cross-linked hyaluronate gel, thecross-linked hyaluronate gel comprising: a cross-linked hyaluronic acidderivative wherein some functional groups of hyaluronic acid arecross-linked to each other with a cross-linking group, the cross-linkedhyaluronic acid derivative having following characteristics: (i) across-linking extent of functional groups of the hyaluronic acid is 1%to 7.5% based on a total number of constituent disaccharide units of thehyaluronic acid, (ii) the cross-linking group is a residue of cinnamicacid or a cinnamic acid derivative, (iii) a degree of substitution of across-linking group is from 10% to 25%, and (iv) a degree ofcross-linking in the cross-linked hyaluronic acid derivative is from 10%to 30%, and a 1% solution of the cross-linked hyaluronate gel is capableof being extruded from a 23-gauge injection needle at 24° C. under apressure of 5.0 Kg/cm².
 2. A solution of a hyaluronic acid derivative ina pharmaceutically acceptable aqueous medium in an amount of 0.1% to 2%by weight based upon the total weight of the solution, the hyaluronicacid derivative comprising cross-linkable groups introduced into part offunctional groups of hyaluronic acid, said hyaluronic acid derivativehaving following characteristics: (i) the cross-linkable groups are aresidue of cinnamic acid or a cinnamic acid derivative, and (ii) adegree of substitution of the cross-linkable groups is from 10% to 25%;and wherein at least one of the following is present: (A) the residue ofcinnamic acid or a cinnamic acid derivative is attached to thefunctional groups of the hyaluronic acid by an amide bond, and whereinthe functional groups are carboxy groups; and (B) the hyaluronic acidderivative has a spacer between the cross-linkable groups and thefunctional groups of the hyaluronic acid.
 3. The solution of ahyaluronic acid derivative according to claim 2, wherein the residue ofcinnamic acid or a cinnamic acid derivative comprises a residue of acinnamic acid derivative in which one or two hydrogen atoms at anypositions of the benzene ring of cinnamic acid are substituted by alower alkyl group having 1 to 8 carbon atoms, a lower alkoxyl grouphaving 1 to 8 carbon atoms, an amino group, or a hydroxyl group.
 4. Thesolution of a hyaluronic acid derivative according to claim 3, whereinthe cinnamic acid derivative comprises aminocinnamic acid orp-aminocinnamic acid.
 5. The solution of a hyaluronic acid derivativeaccording to claim 3, wherein at least the following is present: theresidue of cinnamic acid or a cinnamic acid derivative is attached by anamide bond.
 6. The solution of a hyaluronic acid derivative according toclaim 2, wherein at least the following is present: the residue ofcinnamic acid or a cinnamic acid derivative is attached to thefunctional groups of the hyaluronic acid by an amide bond, and whereinthe functional groups are carboxy groups.
 7. The solution of ahyaluronic acid derivative according to claim 6, wherein the residue ofcinnamic acid or a cinnamic acid derivative comprises a 3-aminopropylcinnamate group.
 8. The solution of a hyaluronic acid derivativeaccording to claim 2, wherein at least the hyaluronic acid derivativehas a spacer between the cross-linkable groups and the functional groupsof the hyaluronic acid.
 9. The solution of a hyaluronic acid derivativeaccording to claim 8, wherein the spacer is a residue of aminoalkylalcohol.
 10. The solution of a hyaluronic acid derivative according toclaim 9, wherein the aminoalkyl alcohol comprises aminopentanol,aminobutanol, aminopropanol, or aminoethanol.
 11. The solution of ahyaluronic acid derivative according to claim 6, wherein the hyaluronicacid derivative is in an amount up to 1% by weight based upon the totalweight of the solution.
 12. The solution of a hyaluronic acid derivativeaccording to claim 1, wherein the hyaluronic acid derivative is in anamount up to about 1% by weight based upon the total weight of thesolution.
 13. The solution of a hyaluronic acid derivative according toclaim 8, wherein the hyaluronic acid derivative is in an amount up to 1%by weight based upon the total weight of the solution.
 14. The solutionof a hyaluronic acid derivative according to claim 2, wherein thehyaluronic acid derivative is in an amount up to about 1% by weightbased upon the total weight of the solution.